EFFECT OF SALIVA AMYLASE ON CHANGES IN PROTEIN PROTEOLYSIS IN THE STOMACH

ABSTRACT

Protein-polysaccharide complexes reduce the rate at which proteins are hydrolysed by gastric juice. At the same time, the α-amylase of saliva can remain active in the stomach for 30 minutes or more. However, the degree of influence of this enzyme on the digestion of a protein-starch mixture in the stomach has not been thoroughly studied, which determined the relevance of this study. The purpose of the study was to evaluate the effect of saliva α-amylase on the activity of gastric proteases during the cleavage of protein-starch substrates. The study was conducted in vitro. The following substrates were used: starch, casein, egg albumin, haemoglobin. Protein solutions were used without the addition of starch, as well as with the addition of starch to protein in a ratio of 1:5 and 5:1. For the casein solution, the total proteolytic activity was 82±7.1 u/ml, for albumin – 69±6.3 u/ml, for haemoglobin – 74±6.8 u/ml. The addition of starch to proteins in a ratio of 1:5 reduced the total proteolytic activity to 51±4.5, 43±3.9, and 46±4.1 u/ml for casein, haemoglobin, and albumin, respectively; in a ratio of 5:1 – to 29±2.7, 20±1.7, and 24±2.2 u/ml (p<0.05). For starch-protein substrates with a ratio of 1:5, exposure to saliva α-amylase for 30 minutes led to pronounced starch hydrolysis and, as a consequence, the resumption of protease activity at the initial level (p>0.05). For starch-protein substrates with a 5:1 ratio, the contact of the substrate with the α-amylase of saliva for 30 minutes was not sufficient for pronounced starch hydrolysis, and the activity of gastric proteases remained reduced (p<0.05). However, the effect of saliva α-amylase on the intensity of proteolysis was still statistically significant compared to the absence of substrate contact with saliva α-amylase (p<0.05). Thus, saliva α-amylase breaks down starch in the composition of protein-starch substrates, which contributes to the activation of proteolysis processes by gastric enzymes. The results obtained can be used in practical medicine to make recommendations on nutrition for patients with endocrinological and gastroenterological pathology.

АННОТАЦИЯ

Белково-полисахаридные комплексы снижают скорость гидролиза белков желудочным соком. В то же время, α-амилаза слюны может сохранять активность в желудке в течение 30 минут и более. Однако степень влияния этого фермента на переваривание белково-крахмальной смеси в желудке изучена недостаточно, что и определило актуальность данного исследования. Цель исследования – оценить влияние α-амилазы слюны на активность желудочных протеаз при расщеплении белково-крахмальных субстратов. Исследование проводилось in vitro. Использовались следующие субстраты: крахмал, казеин, яичный альбумин, гемоглобин. Использовали растворы белков без добавления крахмала, а также с добавлением крахмала к белку в соотношении 1:5 и 5:1. Общая протеолитическая активность для раствора казеина составила 82±7,1 ед/мл, для альбумина – 69±6,3 ед/мл, для гемоглобина – 74±6,8 ед/мл. Добавление крахмала к белкам в соотношении 1:5 снизило общую протеолитическую активность до 51±4,5, 43±3,9 и 46±4,1 ед/мл для казеина, гемоглобина и альбумина соответственно; в соотношении 5:1 – до 29±2,7, 20±1,7 и 24±2,2 ед/мл (p<0,05). Для крахмал-белковых субстратов с соотношением 1:5 воздействие α-амилазы слюны в течение 30 минут приводило к выраженному гидролизу крахмала и, как следствие, возобновлению активности протеаз на исходном уровне (p>0,05). Для крахмал-белковых субстратов с соотношением 5:1 контакт субстрата с α-амилазой слюны в течение 30 минут был недостаточным для выраженного гидролиза крахмала, и активность желудочных протеаз оставалась сниженной (p<0,05). Однако влияние α-амилазы слюны на интенсивность протеолиза всё же было статистически значимым по сравнению с отсутствием контакта субстрата с α-амилазой слюны (p<0,05). Таким образом, α-амилаза слюны расщепляет крахмал в составе белково-крахмальных субстратов, что способствует активации процессов протеолиза ферментами желудка. Полученные результаты могут быть использованы в практической медицине для составления рекомендаций по питанию пациентов с эндокринологической и гастроэнтерологической патологией.

Ключевые слова: белково-полисахаридный комплекс; желудочный сок; гидролиз белка; крахмал; физико-химические характеристики.

Keywords: protein-polysaccharide complex; gastric juice; protein hydrolysis; starch; physicochemical characteristics.

1. Introduction

Proteins and polysaccharides are the main nutrients that enter the body during a standard meal. Differences in environmental factors, such as pH, molecular weight of molecules, ionic strength and charge of proteins, cause the formation of various protein-polysaccharide complexes. In turn, differences in the physicochemical characteristics of protein-polysaccharide complexes determine the degree of stability of food products and their organoleptic qualities (texture, smell, taste). The process of chemical processing of food begins in the oral cavity, where, under the influence of human saliva α-amylase, α-1.4-glycoside bonds of amylose and amylopectin are cleaved. After ingestion, the food lump enters the stomach through the oesophagus, where the proteolysis process begins under the influence of hydrolases (Bornhorst and Singh, 2012). However, in the presence of indigestible salivary polysaccharides, the process of protein breakdown undergoes changes. Starch granules surrounding the protein molecule impair its availability for proteolytic enzymes in gastric juice, which is especially pronounced for gelatinised food during cooking (Palchen et al., 2021). The degree of inhibition of proteolysis directly depends on the molecular weight of polysaccharides, as well as on the protein matrix and the protein-polysaccharide interaction (Duodu et al., 2002). Thus, the presence of indigestible polysaccharides affects the digestion of proteins, but the extent of starch influence and its detailed characteristics have not been studied enough.

The main process of splitting starch occurs in the small intestine. The role of saliva enzymes is considered insignificant, since the short duration of food contact with the α-amylase of saliva in the oral cavity does not allow for full-fledged cleavage of the molecule. In the stomach, saliva α-amylase is inactivated due to changes in the acid-base balance: the pH of saliva ranges from 5.8-7.6, while the pH in the lumen of the stomach body can be only 1.5-2. However, the insignificance of the effect of saliva α-amylase on the cleavage of starch is questioned. D. Freitas et al. (2018) in an in vitro study show that saliva α-amylase in the stomach retains its effectiveness for up to 30 minutes. In previous studies by the authors of this study, it was also confirmed that although the activity of saliva α-amylase decreases at pH 3-5, complete cessation of starch hydrolysis by the enzyme occurs only when pH 2 is reached (Mamajonova et al., 2022).

Thus, the study of the interaction of starch and proteins in the digestive process, as well as the degree of influence of human saliva α-amylase on this process, is an urgent area for research. The relevance of this study is also confirmed by the features of the physiology of nutrition in countries with a hot climate, which includes Uzbekistan. Thus, S.M. Gafarova and L.K. Alimova (2020) describe the need to consume protein in an amount of about 100-110 g/day. A smaller amount of protein does not compensate for the daily nitrogen losses that occur due to increased sweating. A larger amount of protein, in turn, paradoxically leads to an increase in nitrogen losses with sweating. Understanding the processes of protein metabolism depending on the presence of salivary and indigestible polysaccharides is important for making dietary recommendations, especially in people with gastroenterological pathology and in hot climates. This area has not been studied in Uzbekistan, but the influence of proteins and lipids on hydrolysis processes is widely studied. O.K. Jalalova et al. (2019) describe the slowing down of the hydrolysis of fats by pancreatic lipase in the presence of protein molecules (in particular, casein). The researcher note that the presence of fats increases the overall proteolytic activity of pancreatic enzymes and, although to a lesser extent, gastric juice. Consequently, various nutrients can actually act as inhibitors and activators for digestive enzymes.

The purpose of the study was to study the effect of saliva α-amylase on the rate of hydrolysis of proteins that are part of protein-starch complexes.

2. Materials and Methods

This study was carried out in vitro, at the Department of Normal and Pathological Physiology of the Andijan State Medical Institute. The following protein-polysaccharide complexes were used as substrates: a compound of starch and casein; a compound of starch and albumin obtained from eggs; a compound of starch and haemoglobin, as well as protein substrates without starch (casein, egg albumin, haemoglobin). The ratio of starch to proteins in the composition of protein-starch substrates differed and in one case was 1:5, in the other case – 5:1. The duration of incubation with gastric juice without preincubation with saliva for proteins and for protein-starch substrates was 30 minutes. For protein-starch substrates, the method of preincubation with saliva was used for 10 and 30 minutes, followed by incubation with gastric juice for 30 minutes. The following reagents from the manufacturer NeoFroxx (Germany) were used for the study: casein (code 1298, CAS No. 91079-40-2), egg albumin (code 2038, CAS No. 9006-59-1). Soluble starch conforming to GOST 10163-76 standard was also used.

The methods of the study were the determination of the total proteolytic activity (TPA) of gastric juice. The unit of measurement of TPA was the number of units per millilitre (u/ml). The activity of gastric juice proteases was measured using a modified Anson method based on the determination of tyrosine-containing peptides that are released during substrate hydrolysis. Further measurement was carried out by the Lowry method using Folin reagent for colorimetric determination of quantitative protein in solution. Based on the methods used, 2 main research groups were formed: Group 1 – substrates in the ratio of starch to protein 1:5; Group 2 – substrates in the ratio of starch to protein 5:1. Depending on the protein that was part of the protein-starch substrates, Subgroups 1.1 (casein), 1.2 (egg albumin), 1.3 (haemoglobin) and 2.1 (casein), 2.2 (egg albumin), 2.3 (haemoglobin) were formed. Group 4 was a control group in which protein without starch was the substrate, and consisted of Subgroups 3.1 (casein), 3.2 (egg albumin), 3.3 (haemoglobin). All three Subgroups of Group 1 and Group 2 were incubated with gastric juice (30 minutes) and preincubated with saliva for 10 and 30 minutes, followed by incubation with gastric juice (30 minutes). All subgroups of the control group were incubated only with gastric juice for 30 minutes, since they did not contain starch.

Statistical processing and analysis of the obtained results were carried out using the STATISTICA (StatSoft) 12.6 software suite. To check the distribution of the obtained data, the Shapiro-Wilk criterion was applied; for further statistical analysis, parametric statistical methods (arithmetic mean, standard deviation) were used. The difference between the samples was evaluated using the Student-Fisher criterion for two independent samples, using the Wilcoxon criterion – for two consecutive measurements, the differences were evaluated as statistically significant at a significance level of p>0.05. The Microsoft Excel was used to visualise the results obtained.

3. Results

During the measurements, it was found that the TPA of Subgroup 3.1 (casein) was 82±7.1 u/ml, Subgroup 3.2 (albumin) – 69±6.3 u/ml, Subgroups 3.3 (haemoglobin) – 74±6.8 u/ml. These values were used as control values to compare the TPA indicators of Group 1 and 2. The results of the Group 1 TPA measurements are shown in Figure 1.

Figure 1. Levels of total proteolytic activity, Group 1 versus Group 3 (control)

Note: * – significantly different values in relation to the control group.

The level of TPA of Group 1.1 after 30 minutes of incubation with gastric juice (30 minutes) was 51±4.5 u/ml, which is significantly lower compared to TPA of Group 3.1. However, the TPA of Group 1.1 after 10 and 30 minutes of preincubation with saliva with further incubation with gastric juice (30 minutes) was 72 u/ml and 75 u/ml, respectively, which was significantly higher compared to Group 1.1 and did not significantly differ compared to the TPA of Group 3.1. The levels of TPA after 10 and 30-minute exposure to the enzymes contained in saliva did not differ statistically significantly. Thus, the activity of casein hydrolysis by gastric juice significantly decreased with the addition of starch in a ratio of 1:5, but even 10 minutes of preliminary saliva exposure was enough for the activity of casein hydrolysis by gastric juice to reach a level characteristic of the absence of starch.

The level of TPA of Group 1.2 with 30-minute exposure to gastric juice was 43±3.9 u/ml, which is a significantly lower result compared to TPA of Group 3.2. At the same time, the TPA of Group 1.2 after 10 minutes of preincubation with saliva and subsequent 30-minute incubation with gastric juice was 54±4.3 u/ml, which was also significantly lower compared to Group 3.2 and did not differ from the TPA without preincubation. However, the TPA of Group 3.2 after 30-minute preincubation with saliva and 30-minute incubation with gastric juice was 62 u/ml, which was comparable to the indicator of Group 3.2. Consequently, the addition of starch to egg albumin significantly reduced the activity of proteolysis by gastric juice for 30 minutes, regardless of whether preincubation with saliva preceded proteolysis for 10 minutes. However, a 30-minute preincubation of the starch-albumin complex with saliva still allowed to reach a level of TPA comparable to that in the absence of starch.

The level of TPA of Group 1.3 after incubation with gastric juice corresponded to 46±4.1 u/ml, which, as in the previous two cases, was significantly lower compared to the corresponding control group. A 10-minute preincubation with saliva followed by a 30-minute incubation with gastric juice resulted in a statistically significant increase in TPA compared to the complete absence of preincubation. However, this indicator, which was 61 u/ml, still remained significantly lower compared to Group 3.3. Only 30-minute preincubation with saliva provided a TPA of 70 u/ml, which did not differ statistically significantly from the TPA of Group 3.3. Thus, as in the case of albumin, the addition of starch to haemoglobin significantly reduced the activity of proteolysis by gastric juice. The addition of saliva at the preincubation stage for 30 minutes increased the activity of proteolysis significantly, in contrast to the 10-minute preincubation, for which no such effect was observed.

Based on the studies of Group 1 TPA, intermediate conclusions can be drawn. If proteins predominate in the composition of polysaccharide-protein complexes, even a small effect of α-amylase is sufficient for the hydrolysis of 1.4-glycoside bonds. After the carbohydrate bonds are destroyed, the protein-polysaccharide complexes break down, and gastric proteases can contact the substrates without any obstacles. A longer exposure to α-amylase, in this case, will not significantly increase the TPA, since there will be no application point for such an effect in this situation. The results of measuring the TPA of Group 2 are shown in Figure 2.

Figure 2. Levels of total proteolytic activity, Group 2 versus Group 3 (control)

Note: * – significantly different values in relation to the control group.

The level of TPA of Group 2.1. after 30 minutes of incubation with gastric juice without preincubation was 29±2.7 u/ml, which was significantly lower than that of the corresponding control group. After preincubation with saliva for 10 minutes preceding incubation with gastric juice, the level of TPA in Group 2.1 increased slightly and amounted to 40±4.4 u/ml. This indicator did not differ statistically significantly from the indicator typical for incubation with gastric juice without preincubation, and was still significantly lower than the TPA of the control group. Contrary to the trend characteristic of studies in Group 1, preincubation with saliva for 30 minutes also did not allow achieving an indicator that would be comparable to the TPA of the control group – the TPA of Group 2.1 in this case was only 61 u/ml. However, when preincubated with saliva for 30 minutes, the level of TPA was significantly higher compared to preincubation for 10 minutes. Therefore, for protein-starch substrates, which consist of starch and casein in a ratio of 5:1, preincubation with saliva for both 10 and 30 minutes does not allow achieving the level of proteinolysis characteristic of proteins in the absence of starch.

The level of TPA of Group 2.2 during incubation with gastric juice for 30 minutes without preliminary preincubation with saliva was equal to 20±1.7 u/ml. As for Group 2.1, this indicator was significantly lower compared to the TPA of Group 3.2. After the experiment with 10-minute preincubation, the level of TPA corresponding to 30±2.6 u/ml was established, which was also significantly lower than the TPA of the control group and did not differ from the TPA indicator without previous preincubation. Similar to Group 2.1, preincubation for 30 minutes did not allow to reach a level of TPA that would not differ significantly from the control group, but increased TPA to a significantly higher indicator compared to 10-minute preincubation. This indicator for Group 2.1 was 48±3.6 u/ml. Thus, the addition of starch to egg albumin in a ratio of 5:1 significantly reduced the activity of proteolysis. Preincubation with saliva lasting 30 minutes significantly increased TPA, unlike 10-minute preincubation, but the inhibition of proteolysis by starch still remained at a significant level.

For Group 2.3, after incubation with gastric juice for 30 minutes, the TPA level was 24±2.2 u/ml, after incubation with gastric juice for 30 minutes after preincubation with saliva for 10 minutes – 37±4.7 u/ml. In contrast to Groups 2.1 and 2.2, for Group 2.3, 10-minute preincubation with saliva statistically significantly increased TPA compared to only 30-minute incubation with gastric juice. After 30-minute preincubation followed by 30-minute incubation with gastric juice, the TPA of Group 2.3 increased to 53 u/ml. This level of TPA, as in Groups 2.1 and 2.2, was significantly higher compared to TPA after 10 minutes of preincubation, but still was not high enough for comparability with TPA of the control group. Consequently, for the starch-haemoglobin substrate in a ratio of 5:1, even a 10-minute preincubation with saliva was important for increasing the activity of proteolysis, which was not typical for starch-casein and starch-albumin substrate in the same proportions. However, neither 10- nor 30-minute preincubation allowed to completely neutralise the effect of starch on proteolysis activity for Group 2.3.

The main conclusions that characterise Group 2 are a marked decrease in the effectiveness of α-amylase in increasing the activity of proteinolysis. It is possible that if the contact of the protein-starch substrate with α-amylase is prolonged to 60 minutes or longer, the activity of gastric enzymes will reach the initial level. However, such prolonged contact of the substrate with saliva in vivo conditions cannot be guaranteed. A 30-minute exposure to saliva still matters for the activity of protein hydrolysis, probably because some of the starch are still cleaved, thereby ensuring the availability of protein to gastric enzymes. The difference in the hydrolysis activity of casein solution and starch-casein mixture in a ratio of 1:5 after 30 minutes’ incubation with gastric juice and after the previous 10-minute preincubation was 31 u/ml. For the starch-albumin mixture, the difference in TPA was 26 u/ml, and for the starch-haemoglobin mixture – 28 u/ml. In all three studied subgroups, differences in proteolysis activity were statistically significant. The difference in the hydrolysis activity of the casein solution and the starch-casein mixture in a ratio of 5:1 was much more pronounced and amounted to 53 u/ml. For the starch-albumin mixture in the same ratio, this indicator was equal to 49 u/ml, for the starch-haemoglobin mixture – 50 u/ml.

When comparing the activity of proteolysis for Subgroups 1 and 2, the following trend is observed: with more starch in the protein-starch substrate, the same duration of preincubation with saliva has less effect on the activity of gastric proteolysis. This fact confirms that when starch is added to solutions of various proteins, proteins and polysaccharides form complexes whose structure limits the availability of substrates for proteases and thereby makes the process of splitting proteins in their composition more complex and lengthy. The more digestible polysaccharides are included in the complex, the more they limit the possibilities of the effect of gastric enzymes on proteins. It is also necessary to consider the effect of different incubation and preincubation modes on various proteins in the composition of protein-starch polysaccharides. In group 1, 30-minute incubation with gastric juice did not lead to significant differences in TPA between Subgroups 1.1, 1.2, and 1.3. After 10-minute preincubation with saliva, the TPA in Subgroup 1.1 was significantly higher compared to Subgroups 1.2 and 1.3. There were no statistically significant differences between Subgroups 1.2 and 1.3. After 30-minute preincubation with saliva, the TPA in Subgroup 1.2 was significantly lower compared to Subgroup 1.1, but not with Subgroup 1.3. The TPA of Subgroups 1.2 and 1.3 after 30 minutes of preincubation with saliva did not differ from each other.

The above results allow for the conclusion that for casein in the protein-starch substrate, a shorter duration of exposure to saliva α-amylase is sufficient to break down starch and significantly increase the activity of proteolysis compared with albumin and haemoglobin. For albumin, on the contrary, the duration of exposure to saliva α-amylase in order to neutralise the inhibitory effect of starch on protein hydrolysis should be the longest compared to casein and haemoglobin. This trend is typical for the ratio of starch to protein 1:5. In Group 2, there were no statistically significant differences between the TPA of Subgroups 2.1, 2.2, and 2.3 after 30-minute incubation with gastric juice. Preliminary 10-minute preincubation with saliva also did not lead to statistically significant differences between Subgroups 2.1, 2.2, and 2.3. With a preincubation duration of 30 minutes, the TPA of Subgroup 2.1 was significantly higher compared to Subgroups 2.2 and 2.3. Thus, for protein-starch substrates with a 5:1 ratio of starch to protein, a statistically significant increase in TPA was found only for casein after a 30-minute preincubation with saliva, preceding a 30-minute incubation with gastric juice. 10-minute preincubation and its absence for casein, albumin, and haemoglobin resulted in a comparable level of TPA.

4. Discussion

Saliva performs several important functions: it forms and moisturises a food lump, has an antimicrobial effect, protects and restores the mucous membrane of the oral cavity. Hydrolysis of polysaccharides by α-amylase in the oral cavity is important for the prevention of caries. According to D.J. Culp et al. (2021), in the absence of α-amylase in the saliva of mice, the level of Streptococcus mutans in the oral microbiota reached 75-90%. S. mutans is one of the main bacteria that cause tooth decay. Z. Parsaie et al. (2022) show that the probability of developing caries is significantly higher in children with inherently low concentrations of α-amylase in saliva. It can be assumed that the main importance of splitting polysaccharides in the oral cavity is to slow down the growth of bacteria by reducing the amount of sugar available to them. Saliva α-amylase is of great importance for taste recognition. The taste of starch is not recognised by the receptors of the tongue until the cleavage of 1.4-glycoside bonds occurs. According to C. Peyrot des Gachons and P.A.S. Breslin (2016), starch glycolysis activates the early release of insulin, probably after the cleavage of disaccharides to monosaccharides.

However, the effect of α-amylase is not limited to the oral cavity, which is confirmed by this study and other scientific publications. J. Nadia and co-authors consider the traditional approach to be incorrect when modelling gastric digestion takes place in an acidic environment with a pH level <3. The authors demonstrate that in the cardiac part of the stomach, an alkaline environment can persist for up to 60 minutes. At the same time, the longer the pH level>3 is maintained, the smaller the polysaccharide cleavage products are, up to the smallest particles with a size of 2 mm. In addition, J. Nadia et al. (2022) show the importance of different structures and geometries of polysaccharides for their hydrolysis of α-amylase saliva in the stomach: small, porous spherical particles allow faster cleavage, and large granules characteristic of rice and pasta undergo hydrolysis much slower.

D. Freitas and S. Le Feunteun (2018) in an original study demonstrate that the addition to bread or wheat allows the α-amylase of saliva to remain active during the first hour of gastric digestion, resulting in the release of 25-85% starch and 15-50% oligosaccharides. When lemon juice was added to the substrate instead of water, starch hydrolysis decreased by more than 2 times, and the splitting of oligosaccharides did not occur at all. In another study by D. Freitas and S. Le Feunteun (2019) demonstrated that before the inactivation of α-amylase by acidic gastric juice, this saliva enzyme releases up to 80% of bread starch and about 30% of pasta starch, splitting more than half of the long-chain carbohydrates contained in these products to oligosaccharides. Y.A. Mennah-Govela et al. (2015) confirm that the amount of cleavage of polysaccharides by saliva enzymes depends on the time during which the activity of α-amylase in the stomach persists. Moreover, the type of rice influences the intensity of hydrolysis of polysaccharides: the diffusion of acidic gastric juice occurs faster for brown rice, and white rice as part of a food lump retains the alkaline pH in the stomach longer. There are few studies that support the inhibitory effect of polysaccharides on the rate of protein cleavage. Thus, J.O. Markussen et al. (2021) in an in vitro study evaluated the effect of hydrocolloids on the rate of protein breakdown. In particular, the addition of pectin and agar-agar to milk protein concentrate significantly slows down protein hydrolysis.

The above information is fully consistent with the results that were obtained in this study. The effect of α-amylase in the presence of starch remains at a sufficiently active level despite the low pH of gastric juice. The design of this study is unique: the assessment of the total proteolytic activity of gastric juice in the presence of starch and saliva has not been evaluated before, so it is possible to compare the results of this study with other scientific information only indirectly. The results of this study are of practical importance for assessing the glycaemic index of foods, an indicator of the rate of entry of carbohydrates from the digestive system into the blood. Products with a high glycaemic index provoke a rapid increase in insulin in the blood, which, with frequent use of products with a glycaemic index, can lead to insulin resistance and, as a consequence, the development of type 2 diabetes mellitus. The results of this study help to assess the rate of breakdown of polysaccharides in the stomach, which directly determines the glycaemic index of products and can be applied by endocrinologists and nutritionists.

The data obtained in the already mentioned study by Y.A. Mennah-Govela et al. (2015) explain why the glycaemic index of white rice is higher compared to brown rice: white rice allows α-amylase to remain active in the stomach for longer, and brown rice is quickly soaked in gastric juice, which neutralises saliva enzymes. Important for the rate of glycaemic response is not only the amount of long-chain carbohydrates in food, but also the nature of microstructural interactions between proteins, fats, and carbohydrates (Parada and Santos, 2016). On the other hand, T.M.S. Wolever et al. (2021) conclude that the low activity of saliva α-amylase does not lower the glycaemic index of white rice, as does the cold temperature of rice dishes consumed, contrary to the prevailing traditional opinion.

Based on the above data, it can be assumed that the level and activity of saliva α-amylase may be associated with the development of metabolic syndrome and cardiovascular pathology. Nevertheless, the results of studies devoted to this problem are contradictory. According to K. Nakajima (2016) metabolic syndrome, obesity and type 2 diabetes mellitus are the main causes of a decrease in the concentration of α-amylase in saliva. A low level of α-amylase can most likely be associated with the manifestation of diabetes mellitus, which can be caused by both endocrine insufficiencies of the pancreas and insulin resistance. However, A. Ikeda et al. (2021), based on data obtained from in vivo studies, draw the following conclusions: an increase in the level of α-amylase in saliva is associated with higher fasting blood glucose and the development of insulin resistance, diagnosed using the HOMA index (homeostasis model assessment index for insulin resistance) and the results of an oral glucose tolerance test. In addition, with a higher level of α-amylase in saliva, a statistically significant increase in blood pressure and heart rate was observed. The authors of the study explain these effects by an increase in the activity of the sympathetic nervous system, which leads to an increase in the level of alkaline enzymes of saliva. Thus, the practical application of the results obtained in this study and in similar studies requires further analysis and confirmation in in vitro and in vivo experiments.

5. Conclusions

The addition of starch to proteins leads to the formation of protein-starch complexes. Hydrolysis of proteins in such complexes by gastric juice enzymes is much slower compared to the protein substrate. In comparison with substrates that consist exclusively of protein, the addition of starch to protein significantly reduces the activity of proteolysis of the substrate by gastric juice. A significant effect of inhibition of TPA is observed not only at a starch-to-protein ratio of 5:1, but also at lower starch concentrations (ratio 1:5). As the amount of starch in the substrate increases, the activity of proteolysis decreases even more significantly. Thus, the degree of inhibition of proteolysis by polysaccharides directly depends on the amount of starch in the protein-starch mixture.

For protein-starch substrates with a predominance of protein, a 30-minute exposure to amylase returns the activity of their proteolysis by gastric juice to the level characteristic of exclusively protein substrates. This happens both by reducing the amount of starch and facilitating the access of proteases to proteins, and by reducing the total number of protein-starch complexes. Even a 10-minute contact with saliva was enough for the starch-casein substrate to achieve such a result. For those mixtures in which starch predominate, the effectiveness of the effect of α-amylase will be significantly lower. In this case, even after 30 minutes of contact with saliva, the activity of gastric enzymes will remain at a significantly lower level compared to a fully protein substrate, although the effect will be statistically significant compared to the absence of exposure to saliva α-amylase. Since α-amylase remains active in the stomach for up to 60 minutes, a 30-minute contact of this enzyme with a starch-protein substrate is also achieved in vivo.

Thus, the results of this study may be important for understanding the physiology of digestion and such clinical areas of medical science as gastroenterology, endocrinology, and dietetics. An urgent line for further research is to investigate the effect of saliva α-amylase on protein proteolysis in the stomach in various pathological conditions of the digestive system.

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